Mutations Affecting the Development of the Peripheral Nervous System in Drosophila: A Molecular Screen for Novel Proteins

Corresponding author: Sergei N. Prokopenko, The Salk Institute for Biological Studies, MNL-T, P.O. Box 85800, San Diego, CA 92186-5800., prokopenko{at}salk.edu (E-mail)

Communicating editor: T. F. C. MACKAY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

In our quest for novel genes required for the development of the embryonic peripheral nervous system (PNS), we have performed three genetic screens using MAb 22C10 as a marker of terminally differentiated neurons. A total of 66 essential genes required for normal PNS development were identified, including 49 novel genes. To obtain information about the molecular nature of these genes, we decided to complement our genetic screens with a molecular screen. From transposon-tagged mutations identified on the basis of their phenotype in the PNS we selected 31 P-element strains representing 26 complementation groups on the second and third chromosomes to clone and sequence the corresponding genes. We used plasmid rescue to isolate and sequence 51 genomic fragments flanking the sites of these P-element insertions. Database searches using sequences derived from the ends of plasmid rescues allowed us to assign genes to one of four classes: (1) previously characterized genes (11), (2) first mutations in cloned genes (1), (3) P-element insertions in genes that were identified, but not characterized molecularly (1), and (4) novel genes (13). Here, we report the cloning, sequence, Northern analysis, and the embryonic expression pattern of candidate cDNAs for 10 genes: astray, chrowded, dalmatian, gluon, hoi-polloi, melted, pebble, skittles, sticky ch1, and vegetable. This study allows us to draw conclusions about the identity of proteins required for the development of the nervous system in Drosophila and provides an example of a molecular approach to characterize en masse transposon-tagged mutations identified in genetic screens.

THE peripheral nervous system (PNS) of Drosophila has been long used as an experimental paradigm to identify new genes and to further our understanding of the molecular mechanisms of neurogenesis (MODOLELL 1997 ; DAMBLY-CHAUDIERE and VERVOORT 1998 ; JAN and JAN 1998 ). Most known genes that affect PNS development were isolated serendipitously, since they affect easily identifiable morphological markers, namely bristle number in adults (JAN and JAN 1993 ). These genes are often nonessential or correspond to partial loss- or gain-of-function mutations in essential genes. Other players that are required for PNS development are essential genes that were isolated because loss of one gene copy causes a visible, but often unrelated haploinsufficient phenotype (e.g., Notch, Delta, and Enhancer of split; LINDSLEY and ZIMM 1992 ). Later, these genes were shown to affect embryonic neurogenesis when homozygous, and the functional analysis that followed their initial characterization gradually integrated them into developmental pathways of neurogenesis (JAN and JAN 1993 ).

A subset of PNS genes that remained largely unidentified until the late 1980s corresponds to those essential genes that do not cause a haploinsufficient phenotype when mutated. These genes were identified in genetic screens designed to isolate mutations that cause aberrant development of the embryonic PNS (SALZBERG et al. 1994 ; KOLODZIEJ et al. 1995 ; GAO et al. 1999 ). Effects of mutations in these genes are typically pleiotropic and do not affect PNS development only. Two classical examples include the daughterless (CAUDY et al. 1988 ) and numb (UEMURA et al. 1989 ) genes. However, because screening by immunohistochemical staining of fixed whole-mount embryos with monoclonal antibodies is quite tedious (JAN and JAN 1993 ), no such systematic screens were performed prior to 1992.

We set out to screen for genes that are essential and affect PNS development in embryos using chemical agents (SALZBERG et al. 1994 ) and P elements (KANIA et al. 1995 ; SALZBERG et al. 1997 ) as mutagens. Out of a total of 66 genes that affect PNS development, many genes were mapped and some were shown to be allelic to previously characterized genes on the basis of mapping information, similarity of phenotype, and complementation tests. However, numerous genes identified in P-element screens did not seem to correspond to known genes. The most direct approach to determine if these mutations correspond to novel genes and to establish the nature of mutations is to clone the genes adjacent to the P-element insertions. We selected 26 genes for cloning on the basis of several criteria. Combination of plasmid rescue, sequencing, and cDNA cloning gave us molecular information about the mapping position of P elements, their physical locations in the genome, the nature of mutations, the possible identity of encoded proteins, etc. Here, we report on the nature of these mutations and their adjacent genes. We demonstrate that 11 mutations correspond to known genes and report the cloning, sequence, and analysis of expression in the embryo of 10 novel genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Stocks:
All stocks were maintained on a standard corn meal/agar medium (ASHBURNER 1989 ) at room temperature. P{lacZ,w⁺} P-element insertion lines used in this study derive from Istvan Kiss' collection of P elements on the second chromosome (TOROK et al. 1993 ) and from Peter Deák's collection on the third chromosome (SALZBERG et al. 1997 ), and were shown to be associated with phenotypes in the embryonic PNS (KANIA et al. 1995 ; SALZBERG et al. 1997 ). P-element insertion lines used in the screen are listed in Table 2.

Deficiencies and mutations used for complementation tests are listed in Table 1. Deficiencies and mutations were obtained from the Bloomington Stock Center, the Berkeley Drosophila Genome Project, and individual laboratories. Genetic nomenclature, gene names, and cytology are according to LINDSLEY and ZIMM 1992 and FlyBase (flybase.bio.indiana.edu; FLYBASE CONSORTIUM 1999 ).

In situ hybridization to polytene chromosomes:
Digoxigenin-labeled DNA probes were prepared using the DIG DNA labeling kit (Roche Molecular Biochemicals). Pretreatment and hybridization to polytene chromosomes were essentially as described (LANGER-SAFER et al. 1982 ). Following hybridization, probes were detected using an anti-digoxigenin antibody conjugated to alkaline phosphatase (Fab fragments, 1:200; Roche Molecular Biochemicals) and 4-nitroblue tetrazolium chloride with 5-bromo-4-chloro-3-indolyl-phosphate (Roche Molecular Biochemicals). The chromosomes were counterstained with Giemsa (Sigma, St. Louis) and mounted in Permount mounting medium (Fisher Scientific, Pittsburgh, PA).

In situ hybridization to whole-mount embryos:
In situ hybridization to whole-mount Canton-S embryos was carried out as described (TAUTZ and PFEIFLE 1989 ) using digoxigenin-labeled antisense riboprobes (DIG RNA labeling kit; Roche Molecular Biochemicals). To generate riboprobes by run-off transcription, the following combinations of restriction enzymes (to linearize the template plasmid DNA) and RNA polymerases were used: aay antisense probe (5B cDNA, XhoI, T3 polymerase), aay sense (5B cDNA, BamHI, T7 polymerase), dmt antisense (16A cDNA, XhoI, T3 polymerase), dmt sense (16A cDNA, XbaI, T7 polymerase), glu antisense (glu11 cDNA, NotI, T3 polymerase), glu sense (glu11 cDNA, HindIII, T7 polymerase), melt antisense (8G cDNA, XbaI, T7 polymerase or HL03627 cDNA, NotI, T7 polymerase), melt sense (8G cDNA, EcoRV, T3 polymerase or HL03627 cDNA, XhoI, T3 polymerase), stich1 antisense (GM05287 cDNA, XbaI, T7 polymerase), and stich1 sense probe (GM05287 cDNA, XhoI, T3 polymerase).

Molecular biology:
Genomic DNA isolation from Canton-S flies, poly(A)⁺ RNA isolation from 0- to 20-hr-old Canton-S embryos, Southern and Northern analyses, and screening of cDNA libraries were performed according to standard protocols (SAMBROOK et al. 1989 ).

Plasmid rescue:
Genomic sequences flanking the sites of P{lacZ,w⁺} P-element insertions were isolated by plasmid rescue (PIRROTTA 1986 ) using BamHI, XbaI, and PstI (for 5' sequences) and EcoRI and SacII (for 3' sequences) restriction enzymes and Epicurian coli XL1-Blue supercompetent cells (Stratagene, La Jolla, CA). The typical number of transformant colonies with 3 µg of starting genomic DNA and one-third of a ligation reaction used for transformation ranged from 1 to 50.

Several tests were performed on each plasmid rescued genomic fragment to determine if they correspond to novel genes and if they can be used as probes to screen cDNA libraries to clone the corresponding genes. They were (1) checked molecularly by restriction analysis (Table 3), (2) checked cytologically by in situ hybridization to polytene chromosomes (data not shown), (3) analyzed by sequencing (Table 3), and (4) checked on a Southern of Canton-S genomic DNA for the absence of repetitive DNA (data not shown).

For each rescue, at least three colonies were checked by DNA miniprep and restriction analyses. In rare cases, when all three colonies exhibited different digestion patterns, three more colonies were analyzed. The lengths of isolated genomic fragments ranged from 150 bp to 15 kb (see Table 3). In some cases we found upon double digestion (using as a second enzyme XbaI for EcoRI and SacII rescues and HindIII for BamHI, PstI, and XbaI rescues) that a plasmid did not carry a fragment corresponding to a P-element backbone (2 kb for EcoRI and SacII rescues and 10 kb for all other rescues). The presence of a new band that had a size larger or smaller than expected suggested that there were rearrangements of genomic DNA associated with the P-element insertion.

Cytological location of each fragment was verified by in situ hybridization to polytene chromosomes. If a mapping position of a fragment did not correspond to the mapping position of a P-element line used for plasmid rescue, it was discarded.

Based on digestion pattern, a representative plasmid was chosen for sequencing. A single sequencing run was performed (see below). The sequences were used to perform BLAST searches against nucleotide and protein sequence databases. The sequence information from plasmid rescues also provided an independent verification of mapping positions of genomic fragments. If a mapping position of a genomic clone (cosmid, bacterial artificial chromosome, or P1) hit by plasmid rescue-derived sequence was different from a P-element mapping position, the plasmid rescue was excluded from further analysis. Genomic fragments listed in Table 3 have mapping positions identical to P-element lines from which they were derived. BLAST searches also allowed us to determine the origin of genomic fragments for multiple insertion lines (e.g., l(2)k00424). The results of BLAST searches are presented in Table 3.

cDNA cloning:
Plasmid rescue-derived genomic fragments (both 5' and 3', if available) were used to screen cDNA libraries. We used an adult head EXLX M(-) cDNA library (BRUCE A. HAMILTON, personal communication) to isolate 31HC and 31HE clones and an embryonic (9–12 hr) gt11 cDNA library (ZINN et al. 1988 ) to isolate all other cDNA clones (Table 4). For each set of probes, at least 400,000 plaques were screened.

cDNA clones derived from a gt11 library were subcloned into pBluescript II KS(+) (clone glu11) or pBluescript II SK(+) (all other clones) (Stratagene). cDNA clones derived from a EXLX library were converted into pEXLX plasmid clones by Cre-loxP automatic subcloning in vivo (PALAZZOLO et al. 1990 ) using BM25.8 Cre-expressing strain (Novagen).

For some genes, putative cDNA clones were identified through database searches among Drosophila expressed sequence tags (ESTs; RUBIN et al. 2000A ). These are cloned in pBluescript SK(+/-) (clones GM05287, HL03627, and LD13852; Table 4) or in pOT2a (clones GH03082, GH23250, GM14315, and LD47384).

Sequencing:
To determine the terminal sequences of plasmid-rescued genomic fragments the following primers were used: P-ele-R (5'-CGACGGGACCACCTTATGTTATTTC-3') for proximal ends of all rescues; 703 (5'-CGAAAAGTGCCACCTGACGTC-3') for distal ends of EcoRI and SacII rescues; and 1706 (5'-GCCAGCAACGCAAGCTTCTAG-3') for distal ends of BamHI, XbaI, and PstI rescues. To determine full-length sequence of cDNA clones, we used nested deletions generated with an ExoIII/mung bean nuclease deletion kit (Stratagene) in combination with primer walking. Dye primer and dye terminator sequencing (BigDye cycle sequencing ready reaction kits; PE Applied Biosystems, Foster City, CA) was carried out on an ABI Prism 377 DNA sequencer (PE Applied Biosystems). Nucleotide sequences were assembled using an Auto-Assembler (PE Applied Biosystems). All sequences were annotated and deposited in GenBank prior to the end of 1999 (see Table 3 and Table 4 for accession numbers).

Biomolecular search and analysis tools:
Sequence similarity searches were performed using National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/; WHEELER et al. 2000 ) and Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org/blast/) BLAST. Open reading frames (ORFs) were identified with NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/) and codon preference was determined using SeqWeb (Wisconsin Package, Genetics Computer Group). To identify protein domains in predicted amino acid sequences, we used Motif (GenomeNet, Institute for Chemical Research, Kyoto University, Japan; http://www.motif.genome.ad.jp), SMART (EMBL, Heidelberg, Germany; http://smart.embl-heidelberg.de; SCHULTZ et al. 1998 , SCHULTZ et al. 2000 ), and ProfileScan (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland; http://www.isrec.isb-sib.ch/software/PFSCAN_form.html). Other biomolecular tools used were COILS (European Molecular Biology network—Swiss node, http://www.ch.embnet.org/software/COILS_form.html; LUPAS et al. 1991 ) to predict coiled-coil domains, SignalP (Center for Biological Sequence Analysis, The Technical University of Denmark, Lyngby, Denmark; http://www.cbs.dtu.dk/services/SignalP/; NIELSEN et al. 1997 ) to predict the presence and location of signal peptide cleavage sites, TMHMM (Center for Biological Sequence Analysis, The Technical University of Denmark, Lyngby, Denmark; http://www.cbs.dtu.dk/services/TMHMM-1.0/; SONNHAMMER et al. 1998 ) to predict transmembrane helices in proteins, PSORT (University of Tokyo, Tokyo, Japan, http://psort.nibb.ac.jp; NAKAI and HORTON 1999 ) to predict subcellular localization sites of proteins, and PESTfind (Pasteur Institute, Paris, France; http://bioweb.pasteur.fr/seqanal/interfaces/pestfind.html; RECHSTEINER and ROGERS 1996 ) to identify PEST regions.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Rationale of the molecular screen:
To identify novel proteins required for the development of the peripheral nervous system, we decided to clone the affected genes identified in our forward genetic screens (KANIA et al. 1995 ; SALZBERG et al. 1997 ). The basis of the molecular screen (Fig 1) is the observation that P elements often insert in the 5' regions of genes (reviewed by BELLEN 1999 ). Therefore, genomic sequences flanking the sites of P-element insertions often provide information about the identity of genes affected by P elements. Typically, flanking genomic DNA is isolated by plasmid rescue, the 5'- and 3'-ends of rescued fragments are sequenced, and the sequences are used for BLAST searches. Three possible outcomes should be considered. First, P elements may affect known, previously identified genes. Indeed, P-element insertions are often partial loss-of-function mutations that cause mild phenotypes that are quite different from the phenotypes associated with severe loss-of-function alleles (e.g., emc^S009426 and other emc alleles, pbl^S054203; SALZBERG et al. 1997 ). Hence, relying on a similarity of phenotype with genes mapped to the region where the P element maps may not permit making an educated guess as to the identity of a gene. Thus, the molecular information obtained through plasmid rescue should greatly assist in the identification of affected genes. Second, BLAST searches may identify P-element insertions in genes that were cloned, but for which no mutations are available. Finally, P elements for which no significant matches were found in BLAST searches are good candidates for mutations affecting novel genes. Each presumably novel gene/mutation is then characterized using a combination of genetic (complementation tests), cytological (comparison of mapping positions of P elements, plasmid rescued genomic fragments, and candidate allelic genes), and molecular (positioning P-element insertions on the genomic sequence relative to neighboring genes, both known and predicted) approaches.

View larger version (22K): In this window In a new window Download PPT slide   Figure 1. Experimental design of the molecular screen. A general strategy for plasmid rescue using the P{lacZ,w+} P element inserted in the 5'-end of a gene transcription unit is shown. The genomic DNA and rescued genomic fragments are shown in red. For details, see MATERIALS AND METHODS and RESULTS. B, BamHI; E, EcoRI; G, BglII; P, PstI; S, SacII; X, XbaI.

Selection of P-element lines for the screen:
The screen is based on the assumption that the lethality caused by the insertion of a P element maps to the same molecular and cytological region as the P element itself. In other words, the P-element insertion itself, but not some other mutation on the chromosome, causes the observed phenotype. However, previous experience with P-element screens demonstrates that this is not always the case. P-element-encoded transposase is a mutagen (BEALL and RIO 1997 ) that may cause chromosomal breaks resulting in inversions, deletions, and other rearrangements elsewhere on a chromosome. In addition, 2-3 transposase activity may result in the introduction of multiple P elements on a chromosome. Indeed, the screen efficiency defined as the percentage of raw lines that contain a single insertion causing its associated phenotype can be as low as 31% (P-element lines from Kiss' collection; SPRADLING et al. 1999 ). Such considerations precluded us from selecting many lines for the molecular screen (see below).

We, therefore, established the following criteria to select P-element lines for the screen. First, P-element lines have to be single insertion lines and revertible. Second, if the P elements are not revertible, they should fail to complement deficiencies that on the basis of their breakpoints should lack the affected gene. Alternatively, they should fail to complement other independently isolated alleles from the same complementation group that map to the same cytological position as the P element. Third, occasionally a line with multiple insertions can be used to clone the gene. However, this was done only if we were able to demonstrate that only one P-element insertion is responsible for the lethality and phenotype.

A total of 72 novel P-element mutations representing 44 complementation groups were identified in our genetic screens (KANIA et al. 1995 ; SALZBERG et al. 1997 ). All these genes are essential, since P-element insertions cause embryonic, larval, or pupal lethality. Some of the novel genes have been already characterized by others and, hence, are not discussed here—barren (barr, BHAT et al. 1996 ), benchwarmer (bnch; A. KANIA and H. J. BELLEN, unpublished results), bunched (bun; short sighted, shs; TREISMAN et al. 1995 ; DOBENS et al. 1997 , DOBENS et al. 2000 ), gutfeeling (guf, SALZBERG et al. 1996 ; IVANOV et al. 1998 ), homothorax (hth; dorsotonals, dtl; RIECKHOF et al. 1997 ; PAI et al. 1998 ), pavarotti (pav, ADAMS et al. 1998 ), RpL30 (KANIA et al. 1995 ), sanpodo (spdo; DYE et al. 1998 ; SKEATH and DOE 1998 ), schnurri (shn; quo vadis, quo; NUSSLEIN-VOLHARD et al. 1984 ; ARORA et al. 1995 ; GRIEDER et al. 1995 ; STAEHLING-HAMPTON et al. 1995 ), and senseless (sens, NOLO et al. 2000 ). Among the remaining mutations we found 31 P-element strains representing 26 complementation groups (Table 2) that satisfy the required criteria. This number includes four lines selected during an initial stage of the project that were subsequently shown to be allelic to known genes—Star (S, KOLODKIN et al. 1994 ), escargot (esg, WHITELEY et al. 1992 ), extra macrochaetae (emc, ELLIS et al. 1990 ; GARRELL and MODOLELL 1990 ), and string (stg, EDGAR and O'FARRELL 1989 ; JIMENEZ et al. 1990 ) (see Table 2 and SALZBERG et al. 1997 ). We also selected three lines with multiple insertions—l(2)k00424, l(2)k05002, and l(2)k08104. Each of these lines carries two P elements; however, one of them is not responsible for the phenotype, since it either maps to another chromosome or complements deficiencies (Table 2).

The remaining lines described in KANIA et al. 1995 and SALZBERG et al. 1997 were not included in the screen, since they did not satisfy the established selection criteria. Some of them correspond to genes identified by a single nonrevertible P-element insertion, which complements deficiencies uncovering the region of the insertion [e.g., l(2)k05422, l(2)k06712, l(2)k08807, l(3)S136802, misn^S081603]. In other cases, P elements map to cytological intervals with few or no deficiencies [e.g., l(3)S052908]. In addition, absence of independent alleles generated in other P-element or chemical mutagenesis screens did not allow us to genetically map the genes and therefore we were unable to conclude if the P-element insertion is responsible for the phenotype.

Characterization of P-element insertions using flanking genomic DNA sequences:
We used plasmid rescue to recover genomic DNA flanking the insertion sites of 30 P-element lines selected for the screen. For many lines (19 out of 30) we were able to recover DNA flanking both 5'- and 3'-ends of P elements (Table 3). Analysis of genomic sequences flanking P-element insertions provided several types of information. First, a significant sequence match found between a plasmid rescue and cDNA sequence of a known gene demonstrated that this gene is likely to be affected by the P element. Second, availability of genomic sequence information allowed us to physically associate P elements and their flanking genomic fragments with specific sites in the genome. Furthermore, we were able to precisely position the P elements relative to neighboring genes. This information allowed us to make predictions about the identity of genes affected by P elements, about allelic relationships between previously characterized mutations and those identified in our screens, and about other genes adjacent to P elements and linked molecularly to the gene of interest. Results of plasmid rescue experiments including molecular (analysis by gel electrophoresis), sequence (GenBank accession numbers for sequences of ends of plasmid rescued fragments), database (BLASTN and BLASTX search results), and genomic (prediction of P-element locations relative to neighboring genes) analyses are presented in Table 3.

Four classes of genes identified in the screen:
Sequence information derived from plasmid rescues allowed us to assign all genes to one of four classes: (1) previously characterized genes (11 genes), (2) first mutations in cloned genes (1 gene), (3) P-element insertions in genes that were phenotypically characterized, but not identified (1 gene), and (4) novel genes (13 genes).

Previously characterized genes:
Our initial analysis of mutations relied solely on the molecular information derived from genomic DNA flanking the sites of P-element insertions. Using this approach we identified 11 previously characterized genes. We provide both molecular and genetic evidence establishing new allelic relationships between 10 existing mutations (Table 3 and Table 5). We found that Cyclin E (CycE, RICHARDSON et al. 1993 ; KNOBLICH et al. 1994 ) is allelic to fondue (fond, KANIA et al. 1995 ), mindmelt (mm, KANIA et al. 1995 ) is allelic to muscleblind (mbl, BEGEMANN et al. 1997 ; ARTERO et al. 1998 ), patched (ptc, STURTEVANT 1948 ; NUSSLEIN-VOLHARD et al. 1984 ; HOOPER and SCOTT 1989 ; NAKANO et al. 1989 ) is allelic to rubberneck (rubr, KANIA et al. 1995 ), puckered (puc, RING and MARTINEZ ARIAS 1993 ; MARTIN-BLANCO et al. 1998 ) is allelic to hearty (hrt, SALZBERG et al. 1994 , SALZBERG et al. 1997 ), and three rows (thr, NUSSLEIN-VOLHARD et al. 1984 ; D'ANDREA et al. 1993 ; PHILP et al. 1993 ) is allelic to anarchist (anch, KANIA et al. 1995 ). CycE, mbl, and thr are directly affected by P-element insertions fond^k02514, mm^k07103, and anch^k07805b, respectively. In contrast, the rubr^k02507 P element is inserted in the first intron of ptc and the hrt^S023803 P element is inserted in the intron between exons 3 and 4 of puc. Since these introns do not contain any known or predicted genes (ADAMS et al. 2000 ), we considered the possibility that ptc is allelic to rubr and puc is allelic to hrt. In all five cases we were able to demonstrate by complementation tests that the mutations are indeed allelic (Table 2). For example, fond^k05002 insertion complements CycE mutation, but fails to complement another P-element allele, fond^k02514. fond^k02514/CycE⁰⁵²⁰⁶ flies are viable, but adult escapers have rough eyes and wing venation defects indicating that the two mutations are allelic. Similarly, mm^k07103 complements two hypomorphic alleles of mbl (Table 2), but fails to complement mbl^E27, a putative null allele affecting the coding sequence of the gene. There are few mm^k07103/mbl^E27 adult escapers that have wing blisters, wing venation defects, or unexpanded wings. Similarly, hrt^S023803 insertion complements puc¹, but fails to complement puc^A251.1F3 (Table 2), a P-element insertion that affects the same intron as the hrt^S023803 P element. In conclusion, results of complementation tests combined with the sequence data provide strong evidence that the mutations are correctly assigned as allelic to previously identified genes.

Previously, we and others have shown that the l(2)k06921, l(2)k08104, l(3)S024532, and l(3)S058701 mutations are allelic to known genes on the basis of complementation data (BERKELEY DROSOPHILA GENOME PROJECT, unpublished results; SALZBERG et al. 1997 ). Our plasmid rescue and sequence data confirm this allelism for the l(2)k08104 and l(3)S058701 P elements, which are allelic to escargot (esg, WHITELEY et al. 1992 ) and extra macrochaetae (emc, ELLIS et al. 1990 ; GARRELL and MODOLELL 1990 ), respectively, and are inserted in the 5'-untranslated regions (5'-UTRs) of the genes (Table 3). The l(3)S024532 P element is allelic to string (stg, EDGAR and O'FARRELL 1989 ; JIMENEZ et al. 1990 ) and is inserted in the stg gene (Table 2 and Table 3). The l(2)k06921 P-element insertion was shown (BERKELEY DROSOPHILA GENOME PROJECT, unpublished results) to be allelic to Star (S; floater, fltr; KOLODKIN et al. 1994 ) and is inserted 1060 nucleotides (nt) upstream of the S AUG on the (-) strand and 275 nt upstream of the asteroid (ast, HIGSON et al. 1993 ; KOTARSKI et al. 1998 ) AUG on the (+) strand (Table 3) and, therefore, may affect both genes.

cyrano: We and others (BYARS et al. 1999 ) found that cyrano (cyr) is allelic to raw (raw, NUSSLEIN-VOLHARD et al. 1984 ). raw is a dorsal-open group gene required for the regulation of Jun N-terminal kinase (JNK) signaling during dorsal closure that encodes a novel protein of 989 amino acids (aa). We used genomic sequences derived from cyr^k01021 to independently identify through database searches the GH23250 cDNA (RUBIN et al. 2000A ) as a candidate clone for raw/cyr (Table 4). It encodes a new smaller isoform (805 aa) of the RAW protein (Fig 2) generated by alternative splicing and the use of an upstream initiation methionine (BYARS et al. 1999 ). Interestingly, we were unable to align with each other any of the six plasmid rescue sequences derived from cyr^k01021 and cyr^k08801 P elements, suggesting that the two P elements may be far apart. Database analysis revealed that the distance between the two insertions is at least 22 kb. The cyr^k08801 P element is inserted in an intron of the raw gene, and cyr^k01021 is inserted 5 kb upstream of the raw AUG (GenBank accession no. AF186024, BYARS et al. 1999 ), but within the coding sequence of the alternatively spliced GH23250 cDNA.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. Domain structure of novel proteins. Proteins are represented schematically with predicted functional domains and motifs shown in color. Abbreviated names of proteins are indicated on the left, and their lengths (in aa) are indicated on the right. Length of a protein followed by a plus sign indicates that the coding sequence of a cDNA clone is incomplete at the 3'-end. MELT protein does not have any known functional domains. Proteins and the respective domains are drawn to scale. For exact location of domains and motifs refer to respective GenBank accession numbers (Table 4).

unchained: The molecular and genetic analyses of unchained (unch, KANIA et al. 1995 ) suggest that it is allelic to Sin3A (NEUFELD et al. 1998 ; PENNETTA and PAULI 1998 ). Mouse mSin3 is a transcriptional corepressor that forms a ternary complex with the Mad and Max basic helix-loop-helix leucine zipper proteins, recruits other corepressor proteins, and downregulates Myc target genes (AYER et al. 1995 ; SCHREIBER-AGUS and DEPINHO 1998 ). Drosophila embryos homozygous for Sin3A null mutation fail to hatch, but have no obvious defects in muscle or nervous system development (PENNETTA and PAULI 1998 ). We found (Table 3 and data not shown) that the unch^k15501 P element is inserted 24 nt upstream of the the 5'-end of the longest Sin3A mRNA (GenBank accession no. AF024603, NEUFELD et al. 1998 ) or 131 nt upstream of the beginning of the alternatively spliced Sin3A mRNA (GenBank accession no. AJ007518, PENNETTA and PAULI 1998 ). Furthermore, database searches using the B25 genomic fragment flanking unch^k15501 identified the LD13852 clone (RUBIN et al. 2000A ) as a putative unch cDNA (Table 4). It maps to the unch locus (Table 4) and is likely to be affected by the unch^k15501 insertion, which is located 33 nt upstream of its 5'-end (data not shown). The unch^k15501 insertion, although not revertible, fails to complement deficiencies and is therefore likely to be responsible for the unch phenotype (KANIA et al. 1995 ). On the basis of these observations we concluded that unch may be allelic to Sin3A. However, complementation tests between unch^k15501 and several Sin3A alleles (including the Sin3A^ex4 null allele, PENNETTA and PAULI 1998 ) showed that they complement each other (Table 2). Note that the Sin3A⁰⁸²⁶⁹, Sin3A^k05415, and Sin3A^k11222 P elements are inserted in the first intron of unch (PENNETTA and PAULI 1998 ; data not shown), whereas the unch^k15501 P element is inserted 5 kb upstream, in the very beginning of the unch transcription unit (see above). Therefore, the peculiar complementation data may be the result of intragenic complementation, and unch may indeed be allelic to Sin3A. Further phenotypic and genetic analyses will be required to resolve this matter.

The above issues are further complicated as we cannot exclude that the unch^k15501 insertion may affect the amphiphysin gene (RAZZAQ et al. 2000 ), which is transcribed from a complementary strand in the orientation opposite to Sin3A. The unch^k15501 P element is inserted 0.8 kb upstream of the 5'-end of amphiphysin mRNA (Table 3) and may affect the amphiphysin promoter or enhancer elements.

First mutations in cloned genes:
We identified only one P element affecting a gene for which there were no mutations available. Calreticulin (Crc) is the gene mutated by the potp^S114307 P-element insertion. The potp^S114307 P element affects the Crc gene (Table 3), since it is inserted in the 5'-UTR of Crc mRNA, 28 nt upstream of initiator methionine.

Vertebrate calreticulins are major Ca²⁺-binding proteins of endoplasmic reticulum implicated in the regulation of intracellular Ca²⁺ signaling, glycoprotein folding, integrin-mediated cell adhesion, and steroid-dependent gene expression (COPPOLINO et al. 1997 ; KRAUSE and MICHALAK 1997 ). The Drosophila Crc gene (SMITH 1992 ) was previously shown to map to 86B-C (GAMO et al. 1998 ) or 85E1-5 (CHRISTODOULOU et al. 1997 ). The potp^S114307 P element was mapped to 85E (SALZBERG et al. 1997 ). To resolve the ambiguity of the mapping of Crc, we identified through database searches the 1.5-kb LD07621 cDNA clone (RUBIN et al. 2000A ) corresponding to Crc and mapped it to 85E1 (data not shown). These observations, together with the ability to revert the lethality by a precise excision of potp^S114307 (SALZBERG et al. 1997 ), indicate that it is indeed a mutation in the Crc gene.

A Crc mutation was reported to cause hypersensitivity of flies to diethylether anesthesia (Crc^eth-as311, GAMO et al. 1998 ). Our data show that Crc is an essential gene, since the potp^S114307 insertion is homozygous lethal and results in loss of neurons, decreased staining with the MAb 22C10 in the central nervous system (CNS), disorganization of the PNS, and pathfinding defects during embryonic development (SALZBERG et al. 1997 ).

P-element insertions in genes that were identified, but not characterized molecularly:
This class of genes includes those P-element insertions that may serve as cloning tools for previously identified mutations that are not transposon tagged. The pebble (pbl) gene was identified in a chemical mutagenesis screen for mutations affecting the pattern of the embryonic cuticle (JURGENS et al. 1984 ) and is required for cytokinesis during postblastoderm mitoses (HIME and SAINT 1992 ; LEHNER 1992 ). We identified two alleles of pbl in our P-element screen (SALZBERG et al. 1997 ), cloned the gene, and showed that it encodes a putative guanine nucleotide exchange factor for Rho1 G protein (RhoGEF, PROKOPENKO et al. 1999 ).

Novel genes:
We identified 13 novel genes and cloned or identified candidate cDNAs for 10 genes. The identity of the respective proteins, their domain structure, and RNA expression are described in the following sections. The information on cDNA clones including their names, lengths, mapping positions, GenBank accession numbers, and results of Northern analysis and sequence analysis is presented in Table 4 and summarized in Table 5. Cloning and functional characterization of bonus (bon) will be published elsewhere (R. B. BECKSTEAD, S. N. PROKOPENKO and H. J. BELLEN, unpublished results). The identity of three remaining genes remains unknown.

l(2)k00424: The l(2)k00424 strain carries two P-element insertions that were mapped at 30D1-2 and 44F1-2. However, only one insertion (at 44F1-2) is responsible for the lethality and possibly the organizational defects observed in the dorsal cluster of neurons in the PNS (KANIA et al. 1995 ) given the complementation data with deficiencies (Table 2). The genomic sequences flanking this insertion did not show any homologies in database searches. Hence, the l(2)k00424 gene remains unidentified. The second genomic fragment isolated from this strain maps at 30D1-2. This P element is inserted in the 5'-UTR, 21 nt upstream of the initiator methionine of the FKBP59-bp1 gene, which encodes the FK506-binding protein FKBP59, a member of the immunophilin family of proteins (ZAFFRAN 2000 ).

bumper-to-bumper: The bumper-to-bumper (btb) gene was identified by a single revertible P-element insertion that leads to pathfinding and connectivity defects and affects dorsoventral migration of lateral chordotonal neurons (KANIA et al. 1995 ). The P-element insertion in btb^k09901 may affect a predicted gene, CG5380, which encodes a DNA-directed RNA polymerase III. Genomic sequences flanking the 5'-end of the P element show homology to RNA polymerase III subunits from several species (Table 3). This P-element insertion is revertible (KANIA et al. 1995 ) and is located 418 nt upstream of CG5380 AUG. Both P element and a plasmid rescued genomic fragment were mapped to 47A-47B14.

on-the-rack: on-the-rack (rack) was identified by a single revertible P-element insertion that causes loss of LCh5 neurons and affects morphology of neurons in the lateral cluster (KANIA et al. 1995 ). Genomic sequence flanking rack^k15001 P-element insertion did not give any significant matches in database searches (Table 3). Our attempts to clone rack were unsuccessful, because the rack^k15001 P-element insertion is associated with DNA rearrangements affecting the P element (Table 3).

astray: astray (aay) was identified by a single revertible P-element insertion (aay^S042314) that causes severe defects in the axonal trajectories in the embryonic PNS (SALZBERG et al. 1997 ). We cloned the full-length aay cDNA, which encodes a 3-phosphoserine phosphatase (Table 4). L-3-phosphoserine phosphatase (PSPase) catalyzes the last rate-limiting step in the biosynthesis of serine—Mg²⁺-dependent hydrolysis of L-phosphoserine to serine as well as an exchange reaction between L-serine and L-phosphoserine. The AAY protein is homologous to PSPases from mammals, plants, yeast, and bacteria (Table 4 and data not shown) and is most similar to human phosphoserine phosphatase (COLLET et al. 1997 ). AAY and human PSPase belong to a new class of phosphotransferases (COLLET et al. 1998 ) characterized by a conserved N-terminal DXDX(T/V) motif (aa 67–71 in AAY, Fig 2). The first aspartate in this motif is absolutely conserved among all proteins and has been implicated in covalent binding of phosphate and formation of a phosphoenzyme catalytic intermediate (COLLET et al. 1999 ). The two proteins share two other highly conserved motifs (in AAY, aa 155–159 and 203–207, 225–232, Fig 2). Conserved residues within these motifs play an important role in catalysis, as demonstrated by site-directed mutagenesis of other phosphotransferases (P-type ATPases and human PSPase, LINGREL and KUNTZWEILER 1994 ; COLLET et al. 1999 ), and are likely to form a catalytic pocket, as shown by 3-D structure analysis of Pseudomonas haloacid dehalogenase (LI et al. 1998 ).

During embryonic development, ASTRAY is expressed in a complex pattern (Fig 3, A–E). During stage 5 (Fig 3A), ASTRAY is expressed in a highly specific pattern consisting of 7 stripes capped on the dorsal side by a longitudinal stripe. It is also expressed abundantly in the area surrounding the pole cells and the invagination in which the pole cells migrate (Fig 3A and Fig B, arrows). The expression during germ band extension is characterized first by 7 broad stripes (Fig 3B) and later by 10 stripes that eventually fuse to form a peculiar pattern (data not shown). This expression then fades and gives rise to a pattern of segmentally repeated small clusters of cells (Fig 3C, arrowheads), a ring of large cells around the anterior gut (Fig 3, C–E, arrows), and low levels of expression in most of the gut in more mature embryos (Fig 3E).

View larger version (75K): In this window In a new window Download PPT slide   Figure 3. Expression of novel genes during embryonic development. Wild-type embryos were hybridized with aay (A–E), dmt (F–I), glu (J–N), melt (O–S), and stich1 (T–W) antisense RNA probes. Sense probes used in parallel in situ hybridization experiments gave either no staining or a low level of background staining (data not shown). Stages of embryonic development are indicated in each lower right corner. For details on generating RNA probes see MATERIALS AND METHODS.

How does a mutation in PSPase (aay^S042314 P element is inserted in the 5'-UTR of aay) lead to the axon guidance phenotype observed in the PNS? Serine is used not only as a building block for protein synthesis but also as a precursor of phospholipids (phosphatidylserine and sphingomyelin) and glycolipids. Loss of AAY may cause abnormalities in membrane biogenesis in specific cells that would affect transmembrane signaling in neuronal growth cones. Clearly, other alternative hypotheses are possible. It is interesting to note that L-serine does not cross the blood-brain barrier well (SMITH et al. 1987 ) and that defects in the serine biosynthesis pathway leading to low serine levels in cerebrospinal fluid have been described in patients with Williams syndrome (3-phosphoserine phosphatase deficiency, JAEKEN et al. 1997 ) and congenital encephalopathy (3-phosphoglycerate dehydrogenase deficiency, JAEKEN et al. 1996 ).

chrowded: chrowded (chrw) was identified by a single revertible P-element insertion (chrw^k06908) that causes organizational and morphological defects in the PNS (KANIA et al. 1995 ). We identified the LD47384 clone (RUBIN et al. 2000A ) as a candidate chrw cDNA. It encodes a Rab-related small G protein (Fig 2) that is identical to the ORF of the predicted gene CG3870 (RUBIN et al. 2000B ). The chrw^k06908 P element does not directly affect the LD47384 cDNA, but is inserted in close proximity to its 5'-end (Table 3).

CHRW is distantly related to a number of Rab proteins from Drosophila, mammals, plants, and yeast (Table 4 and data not shown), but has a longer ORF (261 aa compared to 200–220 aa in most Rab proteins). Rab proteins compose a separate family within the Ras superfamily that consists of >30 members (in mammals, Rab1–25 and others) implicated in different aspects of intracellular vesicular trafficking (reviewed by ZERIAL and HUBER 1995 ). Rab proteins that share at least 80% identity are included in the same subfamily (e.g., Rab1, Rab2, etc.). In contrast, CHRW shares not more than 40–42% identity (51–54% similarity) with known Rab proteins, including several Rab proteins cloned in Drosophila (SASAMURA et al. 1997 ; SATOH et al. 1997 ). Therefore, we propose that CHRW is a Rab-related protein. Alternatively, it may represent the first member of an unidentified family of Rab proteins. Like other small G proteins, CHRW contains four conserved regions that form the GTP-binding and GTPase catalytic site and the C-terminal CCX cysteine motif as well as the Rab-specific effector region. It is possible that CHRW does not function as a Rab G protein, since it lacks two conserved amino acids present in Rab proteins—G41 in the effector region (A in CHRW) and A151 in region IV (T in CHRW). Further genetic and molecular analyses are necessary to demonstrate that CHRW is indeed a Rab-related protein and that the chrw^k06908 P element affects the Rab gene identified with the LD47384 cDNA.

dalmatian: We identified dalmatian (dmt) in our chemical mutagenesis screen as a mutation that leads to a loss of neurons, disorganization of the PNS, and formation in the ectoderm of small round cells that stain darkly with MAb 22C10 (SALZBERG et al. 1994 ). We used a revertible P-element allele of dmt, dmt^S048103 (SALZBERG et al. 1997 ), to clone the full-length cDNA (Table 4). This cDNA is likely to correspond to the dmt gene, since the dmt^S048103 P element is inserted 11 nt upstream of its 5'-end. dmt encodes a novel protein that is not related to any known or predicted proteins from other species. The only conspicuous feature of DMT is the presence of four nuclear localization sequences (Fig 2). In addition, the PSORT algorithm (NAKAI and HORTON 1999 ) predicts a 78% probability of DMT being a nuclear protein. Analysis of expression pattern by in situ hybridization revealed that at cellular blastoderm, DALMATIAN is expressed at low levels (data not shown). Expression levels increase during germ band extension and the gene is widely expressed at stage 11 (Fig 3F). From stage 12 (Fig 3G), expression becomes more restricted to the PNS and the CNS—ventral nerve cord (Fig 3G and Fig I, arrowheads) and brain (Fig 3, G–I, asterisks). In addition, DALMATIAN is expressed in the pole cells (data not shown), the gut (Fig 3H, arrowheads), and the posterior spiracles (Fig 3H, arrow). In summary, dmt is expressed in numerous tissues including the CNS throughout most of embryonic development and encodes a novel presumably nuclear protein of an unknown function.

gluon: gluon (glu) was identified by a single nonrevertible P-element insertion (glu^k08819) that fails to complement a deficiency uncovering the region where the transposon is inserted (KANIA et al. 1995 ). The embryonic phenotype of glu is characterized by subtle organizational defects in ventral and lateral PNS resulting in irregularly shaped clusters. Sequencing of the genomic fragments flanking glu^k08819 revealed that the P element affects a gene related to SMC (structural maintenance of chromosomes) proteins (Table 3), which are required for chromosome condensation during mitosis (reviewed by STRUNNIKOV 1998 ; HIRANO 1999 ). The nearly full-length glu cDNA encodes a predicted ORF of 1409 amino acids related to members of a highly conserved family of SMC proteins (Table 4)—Xenopus XCAP-C (HIRANO and MITCHISON 1994 ), human CAP-C (NISHIWAKI et al. 1999 ), Saccharomyces cerevisiae Smc4p (KOSHLAND and STRUNNIKOV 1996 ), and Schizosaccharomyces pombe cut3p (SAKA et al. 1994 ). Therefore, GLU is a Drosophila XCAP-C/Smc4-like protein. Similar to other Smc4p-like proteins GLU contains an N-terminal nucleotide-binding motif, two central coiled-coil domains, and a C-terminal DA-box that has DNA-binding capability (Fig 2). The presence of a nuclear localization sequence and PSORT algorithm prediction (91% probability) suggest that GLU is a nuclear protein as demonstrated for XCAP-C (HIRANO and MITCHISON 1994 ) and cut3p (SAKA et al. 1994 ; SUTANI et al. 1999 ).

GLU is likely to be a component of the 13S condensin complex described in Xenopus (HIRANO et al. 1997 ) and S. pombe (SUTANI et al. 1999 ). This complex consists of two SMC subunits (in Xenopus, XCAP-E and XCAP-C) and three non-SMC subunits (in Xenopus, XCAP-D2, XCAP-G, and XCAP-H). 13S condensin as well as its individual components is absolutely required for mitotic chromosome condensation in vitro and in vivo (reviewed by STRUNNIKOV 1998 ; HIRANO 1999 ). Two other components of Drosophila condensin have been identified—Barren (XCAP-H homolog), which is required for sister chromatid segregation during mitosis (BHAT et al. 1996 ), and dSMC2 (XCAP-E homolog, HIRANO 1999 ).

glu has a typical "mitotic" expression pattern similar to other genes implicated in cell cycle regulation or mitosis (e.g., stg, CycA, and barr; EDGAR and O'FARRELL 1989 ; LEHNER and O'FARRELL 1989 , LEHNER and O'FARRELL 1990 ; BHAT et al. 1996 ). Prior to cellularization (Fig 3J), there is maternally provided GLUON RNA that is enriched at the posterior end of the embryo (Fig 3J, arrow), where the pole cells form during telophase of mitotic cycle 10 (stage 4). During germ band extension, there are high levels of GLUON in dividing neuroblasts in the PNS and the CNS (Fig 3K). GLUON expression in the brain (Fig 3, K–N, asterisks) and the ventral nerve cord (Fig 3L and Fig M, arrowheads) persists throughout embryogenesis. By the end of stage 16 (Fig 3N), when most of the embryonic cells have stopped dividing, GLUON RNA expression in most tissues is much lower than at earlier embryonic stages. However, GLUON continues to be expressed at elevated levels in tissues that will resume proliferation during larval development—in neuroblasts in the brain (Fig 3N, asterisk) and in gonads (Fig 3N, arrow). Hence, the expression pattern of GLUON is very similar to BARREN, which encodes another condensin subunit (BHAT et al. 1996 ), but its in vivo role remains to be determined.

hoi-polloi: hoi-polloi (hoip) was identified by a single nonrevertible P-element insertion (hoip^k07104) that fails to complement a deficiency uncovering the region where the transposon is inserted (KANIA et al. 1995 ). hoip^k07104 causes subtle fasciculation and organization defects characterized by misplaced cells within few neuronal clusters. Database searches using genomic sequences flanking the site of hoip^k07104 insertion identified HOIP as a member of a conserved family of YEL026W-like proteins (Table 3). We identified the GH03082 clone (RUBIN et al. 2000A ) as a candidate near full-length hoip cDNA (Table 4). It encodes a 127-aa protein related to human non-histone chromosome protein 2-like 1 (NHP2L1) protein (SAITO et al. 1996 ), Caenorhabditis elegans YEL026W (GenBank accession no. Q21568), and S. cerevisiae Snu13p (GOTTSCHALK et al. 1999 ). These proteins compose an evolutionary conserved family of YEL026W-like proteins found in phyla from plants to humans and distantly related to families of NHP2-like proteins and ribosomal L7Ae proteins (see Table 4 and MAIORANO et al. 1999 ). We propose that HOIP is likely to be a functional homolog of these proteins, since it is very similar to human and C. elegans proteins (79 and 74% identity, respectively; Table 4). HOIP contains a central region corresponding to the ribosomal L7Ae signature (aa 72–89, Motif prediction, see Fig 2) that is highly conserved (83–100% identical) among family members. Human NHP2L1 was shown to bind directly to the 5' stem-loop of U4 small nuclear RNA and is an essential component of a spliceosome (GOTTSCHALK et al. 1999 ). On the basis of these observations we hypothesize that HOIP is an RNA-binding protein, component of a spliceosome, and is required for pre-RNA splicing.

melted: The melted (melt) gene was identified by a single revertible P-element insertion (melt^S144114) that results in abnormal morphology and mild loss of peripheral neurons (SALZBERG et al. 1997 ). We used plasmid-rescued fragments to clone two partially overlapping melt cDNAs (8C and 8G, see Table 4). In addition, through database searches we identified the HL03627 clone (RUBIN et al. 2000A ) as a candidate melt cDNA. We used the full-length sequence of the three clones to assemble a 2.903-kb melt cDNA that is incomplete at the 3'-end. BLAST searches with the sequence of MELT gave no significant results, except a limited homology to a predicted protein from C. elegans (Table 4). In addition, MELT does not contain any functional domains or motifs. Early in embryogenesis (stage 5, Fig 3O), MELTED RNA is expressed in 8 or 9 stripes and in the invaginating ventral furrow (Fig 3O, arrow). During germ band extension, MELTED is expressed in discrete domains in each segment of the embryo (Fig 3P, arrowheads). Later, this pattern is refined to several rows of ectodermal cells in the anterior of each segment (Fig 3, Q–S, arrowheads). There are also low levels of expression in the brain and the gut (data not shown). In conclusion, MELT is a novel protein of unknown function, which, on the basis of its expression pattern, may be required for ectodermal patterning.

skittles: The skittles (sktl) cDNA was isolated in an attempt to clone the fata morgana (fam) gene. fam was identified by several P-element alleles that result in morphological defects of lateral and v' chordotonal neurons in the PNS (KANIA et al. 1995 ). We and others have shown (KNIRR et al. 1997A ; HASSAN et al. 1998 ) that sktl is nested in the first intron of inscuteable (insc; not enough muscles, nem; BURCHARD et al. 1995 ; KRAUT and CAMPOS-ORTEGA 1996 ). fam P-element insertions (e.g., fam^k07505) fail to complement independently generated alleles of insc (Table 2) and affect two genes (KNIRR et al. 1997A ; HASSAN et al. 1998 ). Therefore, the name fata morgana does not refer to either of the two genes, but rather describes the composite phenotype caused by the loss of both. The isolated sktl cDNA (Table 4) encodes a putative phosphatidylinositol-4-phosphate 5-kinase that is longer than the published sequence of SKTL protein (KNIRR et al. 1997B ). SKTL contains a nuclear localization sequence (aa 577–593) and a PEST region (aa 49–78) and has a 78% probability to localize to the nucleus (PSORT prediction). Expression pattern and genetic and functional analyses of sktl have been published elsewhere (KNIRR et al. 1997A ; HASSAN et al. 1998 ).

sticky ch1: The sticky ch1 (stich1) gene was originally identified by a single EMS-induced allele (stich1^D233) as a mutation that affects morphology of neurons (SALZBERG et al. 1994 ). Later, we identified an independently isolated P-element allele of stich1, stich1^S143702 (SALZBERG et al. 1997 ), and showed that the stich1^S143702 insertion is revertible (this study, Table 2). We used one of the genomic sequences flanking the site of the stich1^S143702 insertion to identify through database searches the GM05287 clone (RUBIN et al. 2000A ) as a putative stich1 cDNA. It encodes a predicted basic helix-loop-helix (bHLH) protein similar to Hairy and Enhancer of split-related transcriptional repressors (Table 4, reviewed in FISHER and CAUDY 1998 ). Common functional domains shared among these proteins are the bHLH domain and the adjacent orange domain (Fig 2), which is thought to confer functional specificity among Hairy-related proteins (DAWSON et al. 1995 ). The bHLH domain is most closely related to that of Drosophila HEY, mouse Hey1 (LEIMEISTER et al. 1999 ), and mouse HRT2 (NAKAGAWA et al. 1999 , see Table 4). The basic region is most closely related to that of rat SHARP-1 (ROSSNER et al. 1997 ), human DEC1 (SHEN et al. 1997 ), and mouse Stra13 (BOUDJELAL et al. 1997 ), but the position of a conserved proline residue is shifted in STICH1 2 amino acids toward the N terminus, suggesting that it may have a different DNA-binding specificity. Since the GM05287 cDNA is incomplete at the 3'-end, we do not know if it contains a C-terminal WRPW motif found in all Hairy family proteins and required for interaction with Drosophila non-HLH corepressor protein Groucho (reviewed in FISHER and CAUDY 1998 ). Finally, the predicted protein is much longer (at least 610 aa) than other Hairy and Enhancer of split-related proteins (250–400 aa) and, therefore, it may not be their functional homolog.

The gene has a complex expression pattern during embryonic development (Fig 3, T–W). During stage 8, the RNA is expressed in the anterior and posterior midgut primordia (Fig 3T, asterisks). Expression in the gut continues throughout embryonic development (Fig 3U; hindgut in Fig 3V and Fig W, arrows). During germ band retraction, expression is initiated in many tissues in a prominent segmentally repeated pattern (Fig 3U, arrowheads). Later expression is quite ubiquitous, but has higher levels in segmentally repeated clusters of cells (Fig 3V, arrowheads). Expression is also found in cells of amnioserosa (Fig 3V, asterisk), in the head region (stage 16, Fig 3W), in posterior spiracles (Fig 3W), and in tracheal trees (Fig 3W, arrowheads).

Analysis of the recently released Drosophila genome sequence revealed that the A32 EcoRI and the B32 BamHI genomic fragments (Table 3) are located at least 28 kb apart on the genomic sequence (data not shown). We propose two alternative hypotheses to explain this: (1) they may derive from two different P elements that map 28 kb apart on the stich1^S143702 chromosome or (2) the stich1^S143702 P element may be associated with a 28-kb deletion. We found that the stich1^S143702 P element is inserted 90 nt upstream of the 5'-end of the GM05287 cDNA at 86B. However, the A32 plasmid rescue from stich1^S143702 maps within the first intron of the Domina (Dom) gene, which encodes a transcription factor and maps to 86A2-4 (GenBank accession nos. AJ243814 and AJ243916). This indicates that stich1^S143702 may indeed affect two genes—GM05287 and Domina.

In addition, we identified the EP(3)0359 P element as allelic to stich1, since it fails to complement both stich1^S143702 and stich1^D233 (Table 2 and data not shown). Like stich1^S143702, this EP insertion is located in the first intron of Dom. The close proximity of stich1^S143702 and stich1^EP0359 (the two map 85 bp apart), their failure to complement an independently generated allele of stich1 (stich1^D233, data not shown), and the ability to revert the lethality of stich1^S143702 suggest that stich1 may be allelic to Dom. However, without molecular information on the nature of mutations in stich1^EP0359 and stich1^D233 we cannot exclude the possibility that the stich1^S143702 P element in the intron of Dom is viable and not responsible for the phenotype. Alternatively, both P elements may contribute to the lethality and the PNS phenotype associated with the stich1^S143702 chromosome. We were unable to test these hypotheses, since deficiencies uncovering cytological region 86A-B are not available and there are no recorded Dom mutations. In conclusion, we present identification of a cDNA encoding a bHLH protein similar to a family of Hairy-related transcriptional repressors that may correspond to the sticky ch1 gene.

vegetable: vegetable (veg) was identified by several P-element alleles that cause a severe loss of neurons and fasciculation defects in the PNS (KANIA et al. 1995 ). veg P-element insertions map to 53C or 53E (KANIA et al. 1995 ; SPRADLING et al. 1999 ), leaving the issue of where the veg gene maps unresolved. However, all veg alleles fail to complement each other (KANIA et al. 1995 ). They also fail to complement (Table 2) an independently isolated EMS allele of veg (C. RUSSELL and G. TEAR, personal communication). To clone veg, we selected three lines—veg^k03402, veg^k07202, and veg^k07228. Two of them (veg^k07202 and veg^k07228) carry P elements at 53E1-2 that are revertible (KANIA et al. 1995 and this study) and, therefore, are likely to affect the gene. Since plasmid-rescued fragments from veg^k03402 and veg^k07228 lines carry internal P-element-derived sequences (Table 3 and data not shown), we used the EcoRI plasmid rescue from the veg^k07202 line to clone the gene. We isolated two partially overlapping veg cDNAs (31HC and 31HE, see Table 4) that correspond partially to the sequence (Table 4) of the GM14315 cDNA (RUBIN et al. 2000A ). The assembled nearly full-length 1.809-kb veg cDNA encodes an ORF of 449 aa that is identical to a predicted CG6657 gene product (RUBIN et al. 2000B ). VEG protein has a signal peptide (aa 1–25) and seven transmembrane helices (Fig 2) and is predicted to be a type 3a protein with the N terminus located inside the cell (membrane topology prediction using PSORT, HARTMANN et al. 1989 ). VEG displays similarity to predicted proteins with several transmembrane helices from Arabidopsis, fission yeast, C. elegans, and human (Table 4). Since a P element in the veg^k07202 line is inserted 309 nt upstream of the 5'-end of veg cDNA, it remains to be demonstrated that this P element affects the veg gene. Unexpectedly, we found that veg^k07228 P-element plasmid rescue is 125 kb away from veg^k07202 as well as from the cDNA we cloned, indicating that the veg^k07228 chromosome may carry two P-element insertions or a rearrangement. Since veg^k07202 and veg^k07228 map at 53E1-2 and since the two P elements define genetically the same complementation group and are revertible, we propose that the veg gene maps at 53E1-2. Unfortunately, lack of deficiencies uncovering the cytological divisions 53C-E did not allow us to test this hypothesis. In conclusion, we identified a cDNA that may correspond to the veg gene and that defines a novel family of predicted transmembrane proteins found in organisms from yeast to human.

Candidate cDNAs, novel proteins, and PNS development:
In summary, we present molecular characterization of 26 P-element-tagged mutations and demonstrate that 11 mutations are allelic to previously characterized genes. We identify 13 genes as novel on the basis of genetic and molecular analyses and present cloning of 9 novel genes. At this point the cDNAs we identified should be considered candidate cDNA clones. In many cases P elements directly affect the cDNAs as they are inserted in the coding sequence or in the 5'-UTR (aay, bon, glu, pbl, and raw/cyr) or in close proximity (<100 nt) of the 5'-end of cDNAs (dmt, hoip, and stich1). This strongly suggests that the genes cloned correspond to the respective mutations. However, an mRNA most proximal to the site of a P-element insertion may not be the one or the only one affected by a P element. Known examples include intronic P elements that disrupt a regulatory element of a distal gene without affecting the most proximal gene (e.g., dlt and -Spec, BHAT et al. 1999 ) and P-element insertions affecting two nested genes (e.g., insc and sktl or guf and SmD3, KNIRR et al. 1997A ; HASSAN et al. 1998 ; IVANOV et al. 1998 ). Further molecular, phenotypic, and genetic analyses including transgenic rescue of mutant phenotypes are required to demonstrate if the genes we cloned indeed correspond to the respective mutations.

Results presented here together with our earlier observations based on genetic analyses suggest that there are few genes that function solely in the PNS. Most mutations that affect PNS development are pleiotropic. Such mutations result in phenotypes in other tissues during embryogenesis and probably are required at later stages of development. Indeed, many of the genes identified initially as novel in our screens [e.g., bun (shs), CycE (fond), gcm, hth (dtl), mm (mbl), pbl, ptc (rubr), raw (cyr), S (fltr), shn (quo), thr (anch), and unch (Sin3A)] have been independently isolated in other mutagenesis screens aimed at identifying mutations affecting other cellular or developmental processes. A summary of our current (Table 5) and previous (SALZBERG et al. 1997 ) results demonstrates that mutations in genes required for cell cycle or cell division (e.g., CycA, CycE, glu, pav, pbl, stg, and thr), dorsal closure (e.g., puc and raw), patterning (e.g., ptc), signal transduction (e.g., bun, S, and shn), or cellular metabolism (e.g., aay, btb, guf, hoip, potp, and RpL30) will also affect neuronal development. In conclusion, our results provide information on types of proteins required for the PNS development and may provide a framework for future studies of biochemical and genetic interactions within the molecular pathways operating during nervous system development.

	ACKNOWLEDGMENTS

We thank the Bloomington and Umeå Stock Centers, Istvan Kiss and the Szeged Stock Center, Todd Laverty and the Berkeley Drosophila Genome Project, Bill Chia, Ed Giniger, Corey S. Goodman, Bruce A. Hamilton, Bassem Hassan, Peter Kolodziej, Marek Mlodzik, Daniel Pauli, Giuseppa Pennetta, Claire Russell, Guy Tear, and Kai Zinn for sending us fly strains, antibodies, and cDNA libraries. We acknowledge Bassem Hassan for screening a cDNA library for gluon cDNA and Adi Salzberg for providing us with SacII, PstI, and XbaI plasmid rescues from the l(3)S048103 P-element line. We also thank Nancy Van Driessche for technical assistance; Kwang-Wook Choi (Baylor College of Medicine, Houston, TX), Toru Miki (National Cancer Institute, Bethesda, MD), and Guy Tear (King's College, London, Great Britain) for sharing with us unpublished data; and Anthony L. Lau and Adi Salzberg for helpful discussions. S.N.P. was a graduate student in the Program in Developmental Biology. H.J.B. is an Investigator of the Howard Hughes Medical Institute.

Manuscript received May 5, 2000; Accepted for publication July 27, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

ADAMS, M. D., S. E. CELNIKER, R. A. HOLT, C. A. EVANS, and J. D. GOCAYNE et al., 2000  The genome sequence of Drosophila melanogaster.. Science 287:2185-2195[Abstract/Free Full Text].

ADAMS, R. R., A. A. TAVARES, A. SALZBERG, H. J. BELLEN, and D. M. GLOVER, 1998  pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 12:1483-1494[Abstract/Free Full Text].

ARORA, K., H. DAI, S. G. KAZUKO, J. JAMAL, and M. B. O'CONNOR et al., 1995  The Drosophila schnurri gene acts in the dpp/TGF beta signaling pathway and encodes a transcription factor homologous to the human MBP family. Cell 81:781-790[Medline].

ARTERO, R., A. PROKOP, N. PARICIO, G. BEGEMANN, and I. PUEYO et al., 1998  The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev. Biol. 195:131-143[Medline].

ASHBURNER, M., 1989 Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

AYER, D. E., Q. A. LAWRENCE, and R. N. EISENMAN, 1995  Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80:767-776[Medline].

BAKER, B. S., G. HOFF, T. C. KAUFMAN, M. F. WOLFNER, and T. HAZELRIGG, 1991  The doublesex locus of Drosophila melanogaster and its flanking regions: a cytogenetic analysis. Genetics 127:125-138[Abstract].

BEALL, E. L. and D. C. RIO, 1997  Drosophila P-element transposase is a novel site-specific endonuclease. Genes Dev. 11:2137-2151[Abstract/Free Full Text].

BEGEMANN, G., N. PARICIO, R. ARTERO, I. KISS, and M. PEREZ-ALONSO et al., 1997  muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys₃His-type zinc-finger-containing proteins. Development 124:4321-4331[Abstract].

BELLEN, H. J., 1999  Ten years of enhancer detection: lessons from the fly. Plant Cell 11:2271-2282[Free Full Text].

BERG, C. A. and A. C. SPRADLING, 1991  Studies on the rate and site-specificity of P-element transposition. Genetics 127:515-524[Abstract].

BHAT, M. A., A. V. PHILP, D. M. GLOVER, and H. J. BELLEN, 1996  Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II. Cell 87:1103-1114[Medline].

BHAT, M. A., S. IZADDOOST, Y. LU, K. O. CHO, and K. W. CHOI et al., 1999  Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell 96:833-845[Medline].

BOUDJELAL, M., R. TANEJA, S. MATSUBARA, P. BOUILLET, and P. DOLLE et al., 1997  Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix-loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev. 11:2052-2065[Abstract/Free Full Text].

BREEN, T. R. and I. M. DUNCAN, 1986  Maternal expression of genes that regulate the Bithorax complex of Drosophila melanogaster.. Dev. Biol. 118:442-456[Medline].

BUFF, E., A. CARMENA, S. GISSELBRECHT, F. JIMENEZ, and A. M. MICHELSON, 1998  Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors. Development 125:2075-2086[Abstract].

BURCHARD, S., A. PAULULAT, U. HINZ, and R. RENKAWITZ-POHL, 1995  The mutant not enough muscles (nem) reveals reduction of the Drosophila embryonic muscle pattern. J. Cell Sci. 108:1443-1454[Abstract].

BYARS, C. L., K. L. BATES, and A. LETSOU, 1999  The dorsal-open group gene raw is required for restricted DJNK signaling during dorsal closure. Development 126:4913-4923[Abstract].

CAUDY, M., H. VASSIN, M. BRAND, R. TUMA, and L. Y. JAN et al., 1988  daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex. Cell 55:1061-1067[Medline].

CHEN, Y. and G. STRUHL, 1996  Dual roles for Patched in sequestering and transducing Hedgehog. Cell 87:553-563[Medline].

CHEN, Y. and G. STRUHL, 1998  In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex. Development 125:4943-4948[Abstract].

CHRISTODOULOU, S., A. E. LOCKYER, J. M. FOSTER, J. D. HOHEISEL, and D. B. ROBERTS, 1997  Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue. Gene 191:143-148[Medline].

COLLET, J. F., I. GERIN, M. H. RIDER, M. VEIGA-DA-CUNHA, and E. VAN SCHAFTINGEN, 1997  Human L-3-phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate. FEBS Lett. 408:281-284[Medline].

COLLET, J. F., V. STROOBANT, M. PIRARD, G. DELPIERRE, and E. VAN SCHAFTINGEN, 1998  A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J. Biol. Chem. 273:14107-14112[Abstract/Free Full Text].

COLLET, J. F., V. STROOBANT, and E. VAN SCHAFTINGEN, 1999  Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases. J. Biol. Chem. 274:33985-33990[Abstract/Free Full Text].

COPPOLINO, M. G., M. J. WOODSIDE, N. DEMAUREX, S. GRINSTEIN, and R. ST-ARNAUD et al., 1997  Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386:843-847[Medline].

D'ANDREA, R. J., R. STRATMANN, C. F. LEHNER, U. P. JOHN, and R. SAINT, 1993  The three rows gene of Drosophila melanogaster encodes a novel protein that is required for chromosome disjunction during mitosis. Mol. Biol. Cell 4:1161-1174[Abstract].

DAMBLY-CHAUDIERE, C. and M. VERVOORT, 1998  The bHLH genes in neural development. Int. J. Dev. Biol. 42:269-273[Medline].

DAWSON, S. R., D. L. TURNER, H. WEINTRAUB, and S. M. PARKHURST, 1995  Specificity for the Hairy/Enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol. Cell. Biol. 15:6923-6931[Abstract].

DOBENS, L. L., T. HSU, V. TWOMBLY, W. M. GELBART, and L. A. RAFTERY et al., 1997  The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis. Mech. Dev. 65:197-208[Medline].

DOBENS, L. L., J. S. PETERSON, J. TREISMAN, and L. A. RAFTERY, 2000  Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary. Development 127:745-754[Abstract].

DYE, C. A., J.-K. LEE, R. C. ATKINSON, R. BREWSTER, and P.-L. HAN et al., 1998  The Drosophila sanpodo gene controls sibling cell fate and encodes a tropomodulin homolog, an actin/tropomyosin-associated protein. Development 125:1845-1856[Abstract].

EDGAR, B. A. and P. H. O'FARRELL, 1989  Genetic control of cell division patterns in the Drosophila embryo. Cell 57:177-187[Medline].

ELLIS, H. M., D. R. SPANN, and J. W. POSAKONY, 1990  extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins. Cell 61:27-38[Medline].

FISHER, A. and M. CAUDY, 1998  The function of hairy-related bHLH repressor proteins in cell fate decisions. Bioessays 20:298-306[Medline].

FLYBASE CONSORTIUM,, 1999  The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 27:85-88[Abstract/Free Full Text].

GAMO, S., K. DODO, H. MATAKATSU, and Y. TANAKA, 1998  Molecular genetical analysis of Drosophila ether sensitive mutants. Toxicol. Lett. 100(101):329-337.

GAO, F. B., J. E. BRENMAN, L. Y. JAN, and Y. N. JAN, 1999  Genes regulating dendritic outgrowth, branching, and routing in Drosophila.. Genes Dev. 13:2549-2561[Abstract/Free Full Text].

GARRELL, J. and J. MODOLELL, 1990  The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61:39-48[Medline].

GINIGER, E., K. TIETJE, L. Y. JAN, and Y. N. JAN, 1994  lola encodes a putative transcription factor required for growth and guidance in Drosophila.. Development 120:1385-1398[Abstract].

GOTTSCHALK, A., G. NEUBAUER, J. BANROQUES, M. MANN, and R. LUHRMANN et al., 1999  Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6·U5] tri-snRNP. EMBO J. 18:4535-4548[Medline].

GRIEDER, N. C., D. NELLEN, R. BURKE, K. BASLER, and M. AFFOLTER, 1995  schnurri is required for Drosophila dpp signaling and encodes a zinc finger protein similar to the mammalian transcription factor PRDII-BF1. Cell 81:791-800[Medline].

HARTMANN, E., T. A. RAPOPORT, and H. F. LODISH, 1989  Predicting the orientation of eukaryotic membrane-spanning proteins. Proc. Natl. Acad. Sci. USA 86:5786-5790[Abstract/Free Full Text].

HASSAN, B. A., S. N. PROKOPENKO, S. D. BREUER, B. ZHANG, and A. PAULULAT et al., 1998  skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release. Genetics 150:1527-1537[Abstract/Free Full Text].

HIGSON, T. S., J. E. TESSIATORE, S. A. BENNETT, R. C. DERK, and M. A. KOTARSKI, 1993  The molecular organization of the Star/asteroid region, a region necessary for proper eye development in Drosophila melanogaster.. Genome 36:356-366[Medline].

HIME, G. and R. SAINT, 1992  Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila.. Development 114:165-171[Abstract].

HIRANO, T., 1999  SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates? Genes Dev. 13:11-19[Free Full Text].

HIRANO, T. and T. J. MITCHISON, 1994  A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79:449-458[Medline].

HIRANO, T., R. KOBAYASHI, and M. HIRANO, 1997  Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89:511-521[Medline].

HOOPER, J. E. and M. P. SCOTT, 1989  The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell 59:751-765[Medline].

IVANOV, I. P., K. SIMIN, A. LETSOU, J. F. ATKINS, and R. F. GESTELAND, 1998  The Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP Sm D3 on the opposite strand. Mol. Cell. Biol. 18:1553-1561[Abstract/Free Full Text].

JAEKEN, J., M. DETHEUX, L. VAN MALDERGEM, M. FOULON, and H. CARCHON et al., 1996  3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis. Arch. Dis. Child. 74:542-545[Abstract/Free Full Text].

JAEKEN, J., M. DETHEUX, J. P. FRYNS, J. F. COLLET, and P. ALLIET et al., 1997  Phosphoserine phosphatase deficiency in a patient with Williams syndrome. J. Med. Genet. 34:594-596[Abstract/Free Full Text].

JAN, L. Y., and Y. N. JAN, 1993 The peripheral nervous system, pp. 1207–1244 in The Development of Drosophila melanogaster, edited by M. BATE and A. MARTINEZ ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

JAN, Y. N. and L. Y. JAN, 1998  Asymmetric cell division. Nature 392:775-778[Medline].

JIMENEZ, J., L. ALPHEY, P. NURSE, and D. M. GLOVER, 1990  Complementation of fission yeast cdc2^ts and cdc25^ts mutants identifies two cell cycle genes in Drosophila: a cdc2 homologue and string.. EMBO J. 9:3565-3571[Medline].

JÜRGENS, G., E. WIESCHAUS, C. NÜSSLEIN-VOLHARD, and H. KLUDING, 1984  Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Roux's Arch. Dev. Biol. 193:283-295.

KANIA, A., A. SALZBERG, M. BHAT, D. D'EVELYN, and Y. HE et al., 1995  P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster.. Genetics 139:1663-1678[Abstract].

KLÄMBT, C., J. R. JACOBS, and C. S. GOODMAN, 1991  The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance. Cell 64:801-815[Medline].

KNIRR, S., S. BREUER, A. PAULULAT, and R. RENKAWITZ-POHL, 1997a  Somatic mesoderm differentiation and the development of a subset of pericardial cells depend on the not enough muscles (nem) locus, which contains the inscuteable gene and the intron located gene, skittles.. Mech. Dev. 67:69-81[Medline].

KNIRR, S., A. SANTEL, and R. RENKAWITZ-POHL, 1997b  Expression of the PI4P 5-kinase Drosophila homologue skittles in the germline suggests a role in spermatogenesis and oogenesis. Dev. Genes Evol. 207:127-130.

KNOBLICH, J. A., K. SAUER, L. JONES, H. RICHARDSON, and R. SAINT et al., 1994  Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77:107-120[Medline].

KOLODKIN, A. L., D. J. MATTHES, and C. S. GOODMAN, 1993  The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75:1389-1399[Medline].

KOLODKIN, A. L., A. T. PICKUP, D. M. LIN, C. S. GOODMAN, and U. BANERJEE, 1994  Characterization of Star and its interactions with sevenless and EGF receptor during photoreceptor cell development in Drosophila.. Development 120:1731-1745[Abstract].

KOLODZIEJ, P. A., L. Y. JAN, and Y. N. JAN, 1995  Mutations that affect the length, fasciculation, or ventral orientation of specific sensory axons in the Drosophila embryo. Neuron 15:273-286[Medline].

KOLODZIEJ, P. A., L. C. TIMPE, K. J. MITCHELL, S. R. FRIED, and C. S. GOODMAN et al., 1996  frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 87:197-204[Medline].

KONEV, A. I., E. R. VARENTSOVA, and I. M. KHROMYKH, 1991  Cytogenetic analysis of the chromosomal region containing the radiosensitivity genes of Drosophila. Influence of pericentromeric heterochromatin on mutagenesis in the 44-45 region of chromosome 2. Genetika 27:667-675. [in Russian][Medline].

KOSHLAND, D. and A. STRUNNIKOV, 1996  Mitotic chromosome condensation. Annu. Rev. Cell Dev. Biol. 12:305-333[Medline].

KOTARSKI, M. A., D. A. LEONARD, S. A. BENNETT, C. P. BISHOP, and S. D. WAHN et al., 1998  The Drosophila gene asteroid encodes a novel protein and displays dosage-sensitive interactions with Star and Egfr.. Genome 41:295-302[Medline].

KRAUSE, K. H. and M. MICHALAK, 1997  Calreticulin. Cell 88:439-443[Medline].

KRAUT, R. and J. A. CAMPOS-ORTEGA, 1996  inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. Dev. Biol. 174:65-81[Medline].

LANGER-SAFER, P. R., M. LEVINE, and D. C. WARD, 1982  Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 79:4381-4385[Abstract/Free Full Text].

LEHNER, C. F., 1992  The pebble gene is required for cytokinesis in Drosophila.. J. Cell Sci. 103:1021-1030[Abstract/Free Full Text].

LEHNER, C. F. and P. H. O'FARRELL, 1989  Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell 56:957-968[Medline].

LEHNER, C. F. and P. H. O'FARRELL, 1990  The roles of Drosophila cyclins A and B in mitotic control. Cell 61:535-547[Medline].

LEIMEISTER, C., A. EXTERNBRINK, B. KLAMT, and M. GESSLER, 1999  Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech. Dev. 85:173-177[Medline].

LI, Y. F., Y. HATA, T. FUJII, T. HISANO, and M. NISHIHARA et al., 1998  Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism. J. Biol. Chem. 273:15035-15044[Abstract/Free Full Text].

LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.

LINGREL, J. B. and T. KUNTZWEILER, 1994  Na+,K(+)-ATPase. J. Biol. Chem. 269:19659-19662[Free Full Text].

LUPAS, A., M. VAN DYKE, and J. STOCK, 1991  Predicting coiled coils from protein sequences. Science 252:1162-1164[Free Full Text].

MAIORANO, D., L. J. BRIMAGE, D. LEROY, and S. E. KEARSEY, 1999  Functional conservation and cell cycle localization of the Nhp2 core component of H + ACA snoRNPs in fission and budding yeasts. Exp. Cell Res. 252:165-174[Medline].

MARIGO, V., R. A. DAVEY, Y. ZUO, J. M. CUNNINGHAM, and C. J. TABIN, 1996  Biochemical evidence that Patched is the Hedgehog receptor. Nature 384:176-179[Medline].

MARTÍN-BLANCO, E., A. GAMPEL, J. RING, K. VIRDEE, and N. KIROV et al., 1998  puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila.. Genes Dev. 12:557-570[Abstract/Free Full Text].

MODOLELL, J., 1997  Patterning of the adult peripheral nervous system of Drosophila.. Perspect. Dev. Neurobiol. 4:285-296[Medline].

NAKAGAWA, O., M. NAKAGAWA, J. A. RICHARDSON, E. N. OLSON, and D. SRIVASTAVA, 1999  HRT1, HRT2, and HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic, and pharyngeal arch segments. Dev. Biol. 216:72-84[Medline].

NAKAI, K. and P. HORTON, 1999  PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24:34-35[Medline].

NAKANO, Y., I. GUERRERO, A. HIDALGO, A. TAYLOR, and J. R. WHITTLE et al., 1989  A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched.. Nature 341:508-513[Medline].

NEUFELD, T. P., A. H. TANG, and G. M. RUBIN, 1998  A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148:277-286[Abstract/Free Full Text].

NIELSEN, H., J. ENGELBRECHT, S. BRUNAK, and G. VON HEIJNE, 1997  Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1-6[Abstract/Free Full Text].

NISHIWAKI, T., Y. DAIGO, T. KAWASOE, Y. NAGASAWA, and H. ISHIGURO et al., 1999  Isolation and characterization of a human cDNA homologous to the Xenopus laevis XCAP-C gene belonging to the structural maintenance of chromosomes (SMC) family. J. Hum. Genet. 44:197-202[Medline].

NOLO, R., L. A. ABBOTT, and H. J. BELLEN, 2000  Senseless, a Zn-finger transcription factor is necessary and sufficient for sensory organ development in Drosophila.. Cell 102:349-362[Medline].

NÜSSLEIN-VOLHARD, C., E. WIESCHAUS, and H. KLUDING, 1984  Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193:267-282.

PAI, C. Y., T. S. KUO, T. J. JAW, E. KURANT, and C. T. CHEN et al., 1998  The Homothorax protein activates the nuclear localization of another homeoprotein, Extradenticle, and suppresses eye development in Drosophila.. Genes Dev. 12:435-446[Abstract/Free Full Text].

PALAZZOLO, M. J., B. A. HAMILTON, D. DING, C. H. MARTIN, and D. A. MEAD et al., 1990  Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning. Gene 88:25-36[Medline].

PENNETTA, G. and D. PAULI, 1997  stand still, a Drosophila gene involved in the female germline for proper survival, sex determination and differentiation. Genetics 145:975-987[Abstract].

PENNETTA, G. and D. PAULI, 1998  The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability. Dev. Genes Evol. 208:531-536[Medline].

PHILP, A. V., J. M. AXTON, R. D. SAUNDERS, and D. M. GLOVER, 1993  Mutations in the Drosophila melanogaster gene three rows permit aspects of mitosis to continue in the absence of chromatid segregation. J. Cell Sci. 106:87-98[Abstract].

PIRROTTA, V., 1986 Cloning Drosophila genes, pp. 83–110 in Drosophila: A Practical Approach, edited by D. B. ROBERTS. IRL Press, Oxford.

PROBER, D. A. and B. A. EDGAR, 2000  Ras1 promotes cellular growth in the Drosophila wing. Cell 100:435-446[Medline].

PROKOPENKO, S. N., A. BRUMBY, L. O'KEEFE, L. PRIOR, and Y. HE et al., 1999  A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila.. Genes Dev. 13:2301-2314[Abstract/Free Full Text].

RAZZAQ, A., Y. SU, J. E. MEHREN, K. MIZUGUCHI, and A. P. JACKSON et al., 2000  Characterisation of the gene for Drosophila amphiphysin. Gene 241:167-174[Medline].

RECHSTEINER, M. and S. W. ROGERS, 1996  PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21:267-271[Medline].

RICHARDSON, H. E., L. V. O'KEEFE, S. I. REED, and R. SAINT, 1993  A Drosophila G1-specific cyclin E homolog exhibits different modes of expression during embryogenesis. Development 119:673-690[Abstract].

RIECKHOF, G. E., F. CASARES, H. D. RYOO, M. ABU-SHAAR, and R. S. MANN, 1997  Nuclear translocation of Extradenticle requires Homothorax, which encodes an Extradenticle-related homeodomain protein. Cell 91:171-183[Medline].

RING, J. M. and A. MARTINEZ ARIAS, 1993  puckered, a gene involved in position-specific cell differentiation in the dorsal epidermis of the Drosophila larva. Dev. Suppl. 251–259.

ROCH, F., F. SERRAS, F. J. CIFUENTES, M. COROMINAS, and B. ALSINA et al., 1998  Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs. Mol. Gen. Genet. 257:103-112[Medline].

RØRTH, P., K. SZABO, A. BAILEY, T. LAVERTY, and J. REHM et al., 1998  Systematic gain-of-function genetics in Drosophila.. Development 125:1049-1057[Abstract].

ROSSNER, M. J., J. DORR, P. GASS, M. H. SCHWAB, and K. A. NAVE, 1997  SHARPs: mammalian Enhancer-of-Split- and Hairy-related proteins coupled to neuronal stimulation. Mol. Cell. Neurosci. 9:460-475[Medline].

RUBIN, G. M., L. HONG, P. BROKSTEIN, M. EVANS-HOLM, and E. FRISE et al., 2000a  A Drosophila complementary DNA resource. Science 287:2222-2224[Abstract/Free Full Text].

RUBIN, G. M., M. D. YANDELL, J. R. WORTMAN, G. L. GABOR MIKLOS, and C. R. NELSON et al., 2000b  Comparative genomics of the eukaryotes. Science 287:2204-2215[Abstract/Free Full Text].

SAITO, H., T. FUJIWARA, S. SHIN, K. OKUI, and Y. NAKAMURA, 1996  Cloning and mapping of a human novel cDNA (NHP2L1) that encodes a protein highly homologous to yeast nuclear protein NHP2. Cytogenet. Cell Genet. 72:191-193[Medline].

SAKA, Y., T. SUTANI, Y. YAMASHITA, S. SAITOH, and M. TAKEUCHI et al., 1994  Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J. 13:4938-4952[Medline].

SALZBERG, A., D. D'EVELYN, K. L. SCHULZE, J.-K. LEE, and D. STRUMPF et al., 1994  Mutations affecting the pattern of the PNS in Drosophila reveal novel aspects of neuronal development. Neuron 13:269-287[Medline].

SALZBERG, A., K. GOLDEN, R. BODMER, and H. J. BELLEN, 1996  gutfeeling, a Drosophila gene encoding an antizyme-like protein, is required for late differentiation of neurons and muscles. Genetics 144:183-196[Abstract].

SALZBERG, A., S. N. PROKOPENKO, Y. HE, P. TSAI, and M. PÁL et al., 1997  P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development. Genetics 147:1723-1741[Abstract].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SASAMURA, T., T. KOBAYASHI, S. KOJIMA, H. QADOTA, and Y. OHYA et al., 1997  Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families. Mol. Gen. Genet. 254:486-494[Medline].

SATOH, A. K., F. TOKUNAGA, and K. OZAKI, 1997  Rab proteins of Drosophila melanogaster: novel members of the Rab-protein family. FEBS Lett. 404:65-69[Medline].

SCHREIBER-AGUS, N. and R. A. DEPINHO, 1998  Repression by the Mad(Mxi1)-Sin3 complex. Bioessays 20:808-818[Medline].

SCHULTZ, J., F. MILPETZ, P. BORK, and C. P. PONTING, 1998  SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA 95:5857-5864[Abstract/Free Full Text].

SCHULTZ, J., R. R. COPLEY, T. DOERKS, C. P. PONTING, and P. BORK, 2000  SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 28:231-234[Abstract/Free Full Text].

SHEN, M., T. KAWAMOTO, W. YAN, K. NAKAMASU, and M. TAMAGAMI et al., 1997  Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes. Biochem. Biophys. Res. Commun. 236:294-298[Medline].

SKEATH, J. B. and C. Q. DOE, 1998  Sanpodo and Notch act in opposition to Numb to distinguish sibling neuron fates in the Drosophila CNS. Development 125:1857-1865[Abstract].

SMITH, M. J., 1992  Nucleotide sequence of a Drosophila melanogaster gene encoding a calreticulin homologue. DNA Seq. 3:247-250[Medline].

SMITH, Q. R., S. MOMMA, M. AOYAGI, and S. I. RAPOPORT, 1987  Kinetics of neutral amino acid transport across the blood-brain barrier. J. Neurochem. 49:1651-1658[Medline].

SONNHAMMER, E. L. L., G. VON HEIJNE and A. KROGH, 1998 A hidden Markov model for predicting transmembrane helices in protein sequences, pp. 175–182 in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, edited by J. GLASGOW. AAAI Press.

SPRADLING, A. C., D. STERN, I. KISS, J. ROOTE, and T. LAVERTY et al., 1995  Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc. Natl. Acad. Sci. USA 92:10824-10830[Abstract/Free Full Text].

SPRADLING, A. C., D. STERN, A. BEATON, E. J. RHEM, and T. LAVERTY et al., 1999  The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153:135-177[Abstract/Free Full Text].

STAEHLING-HAMPTON, K., A. S. LAUGHON, and F. M. HOFFMANN, 1995  A Drosophila protein related to the human zinc finger transcription factor PRDII/MBPI/HIV-EP1 is required for dpp signaling. Development 121:3393-3403[Abstract].

STRUNNIKOV, A. V., 1998  SMC proteins and chromosome structure. Trends Cell Biol. 8:454-459[Medline].

STURTEVANT, A. H., 1948  New mutants report. Dros. Inf. Serv. 22:55-56.

SU, T. T. and P. H. O'FARRELL, 1998  Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles. J. Cell Biol. 140:451-460[Abstract/Free Full Text].

SUTANI, T., T. YUASA, T. TOMONAGA, N. DOHMAE, and K. TAKIO et al., 1999  Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4. Genes Dev. 13:2271-2283[Abstract/Free Full Text].

TAUTZ, D. and C. PFEIFLE, 1989  A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.. Chromosoma 98:81-85[Medline].

TEARLE, R. G. and C. NÜSSLEIN-VOLHARD, 1987  Tübingen mutants and stock list. Dros. Inf. Serv. 66:209-269.

TÖRÖK, T., G. TICK, M. ALVARADO, and I. KISS, 1993  P-lacW insertional mutagenesis on the second chromosome of Drosophila melanogaster: isolation of lethals with different overgrowth phenotypes. Genetics 135:71-80[Abstract].

TREISMAN, J. E., Z. C. LAI, and G. M. RUBIN, 1995  shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-beta-responsive gene. Development 121:2835-2845[Abstract].

UEMURA, T., S. SHEPHERD, L. ACKERMAN, L. Y. JAN, and Y. N. JAN, 1989  numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58:349-360[Medline].

WHEELER, D. L., C. CHAPPEY, A. E. LASH, D. D. LEIPE, and T. L. MADDEN et al., 2000  Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 28:10-14[Abstract/Free Full Text].

WHITELEY, M., P. D. NOGUCHI, S. M. SENSABAUGH, W. F. ODENWALD, and J. A. KASSIS, 1992  The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes. Mech. Dev. 36:117-127[Medline].

ZAFFRAN, S., 2000  Molecular cloning and embryonic expression of dFKBP59, a novel Drosophila FK506-binding protein. Gene 246:103-109[Medline].

ZERIAL, M., and L. A. HUBER (Editors), 1995 Guidebook to the Small GTPases. Oxford University Press, Oxford.

ZHANG, N., J. ZHANG, Y. CHENG, and K. HOWARD, 1996  Identification and genetic analysis of wunen, a gene guiding Drosophila melanogaster germ cell migration. Genetics 143:1231-1241[Abstract].

ZINN, K., L. MCALLISTER, and C. S. GOODMAN, 1988  Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila. Cell 58:577-587.

This article has been cited by other articles:

				A. L. Franciscovich, A. D. V. Mortimer, A. A. Freeman, J. Gu, and S. Sanyal Overexpression Screen in Drosophila Identifies Neuronal Roles of GSK-3{beta}/shaggy as a Regulator of AP-1-Dependent Developmental Plasticity Genetics, December 1, 2008; 180(4): 2057 - 2071. [Abstract] [Full Text] [PDF]

				S. T. Harbison and A. Sehgal Quantitative Genetic Analysis of Sleep in Drosophila melanogaster Genetics, April 1, 2008; 178(4): 2341 - 2360. [Abstract] [Full Text] [PDF]

				L. Nicholson, G. K. Singh, T. Osterwalder, G. W. Roman, R. L. Davis, and H. Keshishian Spatial and Temporal Control of Gene Expression in Drosophila Using the Inducible GeneSwitch GAL4 System. I. Screen for Larval Nervous System Drivers Genetics, January 1, 2008; 178(1): 215 - 234. [Abstract] [Full Text] [PDF]

				K. Koizumi, H. Higashida, S. Yoo, M. S. Islam, A. I. Ivanov, V. Guo, P. Pozzi, S.-H. Yu, A. C. Rovescalli, D. Tang, et al. RNA interference screen to identify genes required for Drosophila embryonic nervous system development PNAS, March 27, 2007; 104(13): 5626 - 5631. [Abstract] [Full Text] [PDF]

				S. H. Eun, K. Lea, E. Overstreet, S. Stevens, J.-H. Lee, and J. A. Fischer Identification of Genes That Interact With Drosophila liquid facets Genetics, March 1, 2007; 175(3): 1163 - 1174. [Abstract] [Full Text] [PDF]

				D. Sambandan, A. Yamamoto, J.-J. Fanara, T. F. C. Mackay, and R. R. H. Anholt Dynamic Genetic Interactions Determine Odor-Guided Behavior in Drosophila melanogaster Genetics, November 1, 2006; 174(3): 1349 - 1363. [Abstract] [Full Text] [PDF]

				L. T. Reiter, T. N. Seagroves, M. Bowers, and E. Bier Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase Hum. Mol. Genet., September 15, 2006; 15(18): 2825 - 2835. [Abstract] [Full Text] [PDF]

				T. F. C. Mackay, R. F. Lyman, and F. Lawrence Polygenic Mutation in Drosophila melanogaster: Mapping Spontaneous Mutations Affecting Sensory Bristle Number Genetics, August 1, 2005; 170(4): 1723 - 1735. [Abstract] [Full Text] [PDF]

				M. J. Laviolette, P. Nunes, J.-B. Peyre, T. Aigaki, and B. A. Stewart A Genetic Screen for Suppressors of Drosophila NSF2 Neuromuscular Junction Overgrowth Genetics, June 1, 2005; 170(2): 779 - 792. [Abstract] [Full Text] [PDF]

				L. Michaut, S. Flister, M. Neeb, K. P. White, U. Certa, and W. J. Gehring Analysis of the eye developmental pathway in Drosophila using DNA microarrays PNAS, April 1, 2003; 100(7): 4024 - 4029. [Abstract] [Full Text] [PDF]

				B. Egger, R. Leemans, T. Loop, L. Kammermeier, Y. Fan, T. Radimerski, M. C. Strahm, U. Certa, and H. Reichert Gliogenesis in Drosophila: genome-wide analysis of downstream genes of glial cells missing in the embryonic nervous system Development, March 9, 2003; 129(14): 3295 - 3309. [Abstract] [Full Text] [PDF]

				R. Dubruille, A. Laurencon, C. Vandaele, E. Shishido, M. Coulon-Bublex, P. Swoboda, P. Couble, M. Kernan, and B. Durand Drosophila Regulatory factor X is necessary for ciliated sensory neuron differentiation Development, January 12, 2002; 129(23): 5487 - 5498. [Abstract] [Full Text] [PDF]